Methods and devices for thawing frozen biological samples

ABSTRACT

Methods of thawing frozen biological samples are provided. Aspects of embodiments of the methods include thawing of a biological sample under pressure in a manner sufficient to maintain the structural integrity and/or viability of the sample. Also provided are devices and systems for use in practicing the methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is filed concurrently with the application entitled “Methods and Devices for Imaging and Manipulating a Biological Sample, Attorney Docket Number BIOT-009US2, and claims the priority benefit of U.S. provisional application No. 60/789,541, filed Apr. 4, 2006, which applications are incorporated herein by reference in their entirety.

BACKGROUND

The development of microscopy has allowed scientists to image cells and tissues with increasing levels of detail. This has not only increased our understanding of the structure and composition of various biological tissues and cells but has advanced the development of new protocols for the investigation, screening and diagnosis of disease. Foundational to this advancement has been the development and use of the electron microscope. The electron microscope uses electron bombardment to magnify a sample. Using electron microscopy, biological cells may be imaged at high resolution and magnified over 2 million times. This allows for the imaging of cells and their structures in very minute detail.

There are several ways in which a cell sample can be prepared for imaging by an electron microscope. For instance, the cells can be fixed so as to preserve the sample. Specifically, the sample may be dehydrated and the water in the cell sample replaced with an organic solvent, such as ethanol or acetone. The organic solvent may then be replaced with a plastic medium such as a resin (e.g., araldite or epoxy) to fix the fine structure of the sample. The sample may also be stained, for instance, by a heavy metal or dye, so as to generate better contrast between cellular components. Additionally, the sample embedded with a resin may be sectioned to give very thin slices that can then be separately imaged.

Recently, cryofixation has been used to rapidly freeze a cell sample so as to fix the sample and preserve the cells in their natural state. Once frozen, embedded in resin, stained and sectioned, the cell sample can then be viewed by electron microscopy.

SUMMARY

Methods of thawing frozen biological samples are provided. Aspects of embodiments of the methods include thawing a biological sample under pressure in a manner sufficient to maintain the structural integrity and/or viability of the sample. Also provided are devices and systems for use in practicing the methods.

In certain embodiments, the methods include thawing a biological sample, wherein a previously frozen biological sample is thawed under pressure in a manner sufficient to maintain the structural integrity of the sample. For instance, in certain embodiments, the methods include the thawing of a biological sample under high pressure.

In other embodiments, the methods include thawing a viable biological sample in a manner sufficient to maintain the viability of the biological sample. For instance, in certain embodiments the methods include the high pressure thawing of a viable biological sample wherein the sample has not previously been fixed, stained, embedded, or otherwise treated in a manner that destroys the viability of the biological sample. In certain other embodiments, the methods include ambient pressure thawing a viable biological sample, frozen under high pressure, in a manner sufficient to maintain the viability of the biological sample.

Additionally, in certain embodiments, the methods include freezing or refreezing a biological sample in a manner sufficient to prevent or at least reduce the formation of ice crystals generated by the freezing process. In certain embodiments, the methods include freezing a sample under high pressure wherein the sample has previously been thawed.

In other embodiments, the methods include freezing or refreezing a viable biological sample in a manner sufficient to maintain the viability of the biological sample. For instance, in certain embodiments, the methods include high pressure freezing of a previously thawed viable biological sample wherein the sample has not previously been fixed, stained, embedded, or otherwise treated in a manner that destroys the viability of the biological sample.

The methods of the invention may be used for reversibly and repeatedly thawing and freezing (e.g., cryofixing) a viable biological sample under pressure, for instance, under high pressure. Additionally, once frozen the biological sample may be stored over a prolonged period of time and then thawed, or repeatedly frozen and thawed, while maintaining the viability of the biological sample.

Also provided is an apparatus for performing the methods of the invention. An apparatus of the invention is configured for freezing and thawing a sample, for instance, a biological sample, under pressure (e.g., high pressure). In certain embodiments, the apparatus includes a chamber that includes an interior configured for holding a sample, a pressure modulator for modulating the pressure within the interior of the chamber and a temperature modulator for modulating the temperature of the interior of the chamber from a temperature that is below the freezing point of water to a temperature that is above the freezing point of water and vice-versa.

Accordingly, in certain embodiments, an apparatus of the invention is configured for both generating a high pressure within a chamber and for rapidly transferring heat into and out of the chamber and thereby thawing or heating or freezing or cooling a sample held therein. In certain embodiments, the apparatus includes a plurality of anvils, a pressure modulator, a pressure chamber and/or sample holding element, and a temperature modulator. In accordance with these embodiments, the anvils are configured for being compressed and thereby generating a high pressure within the pressure chamber. The pressure chamber may include a sample holding element, for instance, a gasket, which is configured for holding a sample and is adapted for withstanding the high pressure generated by the compression of the anvils. The pressure modulator, for instance, one or more levers, is configured for compressing the anvils so as to generate a high pressure and the temperature modulator, for instance, an applied gas or liquid, is configured for contacting the anvils and thereby modulating the temperature within the chamber.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates one embodiment of an apparatus of the invention.

FIG. 2 illustrates another embodiment of an apparatus of the invention.

FIGS. 3 through 5 are cross section views of the predicted temperature distribution inside a sample cell of an apparatus of the invention, at various times during the cooling down of the sample.

FIGS. 6 through 8 are cross section views of the predicted temperature distribution inside a sample cell of an apparatus of the invention, at various times during the warming up of the sample.

FIG. 9 shows the predicted temperature at the center of the sample, during cooling down, inside an apparatus of the invention.

FIG. 10 shows the predicted temperature at the center of the sample, during warming up, inside an apparatus of the invention.

FIG. 11 is a slide of a tissue sample (control) that was neither pressurized nor frozen. Rat brain was kept in ice cold bicarbonated Hextend for 15 minutes, cut into 200 μm thick slices, then stained with Molecular Probes L-3224 live/dead stain, and imaged with Zeiss Meta 510 Confocal Laser Scanning Microscope. The L-3224 Live/Dead stain stains the cytoplasm of living cells green and the nuclei of dead cells red.

FIG. 12 is a slide of a tissue sample that was pressurized but was not frozen. Rat brain was kept in ice cold bicarbonated Hextend, pressurized to 2177 atm for 5 minutes, cut into 200 μm thick slices, then stained with Molecular Probes L-3224 live/dead stain and imaged with Zeiss Meta 510 Confocal Laser Scanning Microscope.

FIG. 13 is a slide of a tissue sample that was frozen and thawed while pressurized. Rat brain was kept in ice cold bicarbonated Hextend, then pressurized to 2177 atm, frozen to −196° C. over 5 minutes, held at this temperature and pressure for 5 minutes, then thawed over 15 minutes while held at 2177 atm pressure, then depressurized, cut into 200 μm thick slices, stained with Molecular Probes L-3224 live/dead stain, and imaged with Zeiss Meta 510 Confocal LSM.

FIGS. 14 and 15 are slides of a tissue sample that was frozen quickly at 2100 atm pressure then thawed at 1 atm. Rat brain was cut into 200 μm thick slices, then soaked in ice cold bicarbonated Hextend +20% glycerol, then rapidly frozen under 2100 atm pressure to −196° C. in a Bal-Tec HPM-010 machine, then thawed at 1 atm pressure by immersion in Hextend at 20° C., stained with Molecular Probes L-3224 live/dead stain, and imaged with Zeiss Meta 510 Confocal Laser Scanning Microscope.

DETAILED DESCRIPTION

Methods of thawing frozen biological samples are provided. Aspects of embodiments of the methods include thawing a biological sample under pressure in a manner sufficient to maintain the structural integrity and/or viability of the sample. Also provided are devices and systems for use in practicing the methods.

Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the stated ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference in their entirety as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

As summarized above, embodiments of the methods of the invention are directed to preparing a sample. An aspect of embodiments of the methods includes thawing a sample, for instance, a biological sample, under pressure in a manner sufficient to maintain the structural integrity of the biological sample. In certain instances, the biological sample is a viable biological sample and the methods of the invention include thawing the sample in a manner sufficient to maintain the viability of the biological sample.

Accordingly, in certain embodiments, the methods of the invention involve the high pressure thawing of a frozen sample. By “thawing” is meant warming a sample so as to convert the frozen material therein to a non-frozen state. In certain embodiments, thawing of the sample is achieved by changing the temperature of the sample from a first temperature to a second temperature. In certain embodiments, the first temperature ranges from about 0 K to about 280 K, such as from about 40 K to about 273 K, including about 77 K to about 200 K and the second temperature ranges from about 250 K to about 350 K, such as from about 274 K to about 310 K, including about 288 K to about 300 K or about 298 K. Thawing of the sample may be acheived using any convenient protocol, e.g., via application of heat to the sample, where heat may be applied to the sample, for instance, by the specific protocols detailed below. Although the thawing can take place over a prolonged period of time, in certain embodiments, it occurs rapidly. For instance, in certain embodiments the thawing has a duration ranging from about 1 millisecond to about 1 second, from about 3 milliseconds to about 30 milliseconds, such as from about 5 milliseconds to about 20 milliseconds and including from about 7 to about 15 milliseconds, e.g., about 10 milliseconds.

As summarized above, thawing occurs under high pressure. By “high pressure” is meant a pressure in the range of about 200 atm to about 4000 atm, for instance, about 1800 atm to about 2800 atm, or about 2000 atm to about 2200 atm.

Thawing can take place either with or without the addition of agents that suppress the formation of ice crystals. Ice crystal formation deterrent agents of interest include, but are not limited to: glycerol, dimethyl sulfoxide, polyvinyl pyrridole, sucrose, and the like. Other ice crystal deterrent agents of interest include antifreeze proteins and antifreeze glycoproteins and other freeze protecting compounds found naturally in certain cold tolerant organisms, such as certain arctic and/or antarctic fishes and insects, and synthetic versions or derivatives or variants of such proteins and glycoproteins and other naturally occurring freeze protecting compounds found in organisms that live in cold environments. Combinations of such agents may also be used. However, although thawing can take place with the addition of agents that suppress the formation of crystals during the thawing process, in certain embodiments, the thawing process occurs without the addition of such agents.

In certain embodiments, the methods of the invention also include the freezing or refreezing of a sample under pressure, for instance, a high pressure. By “high pressure freezing” is meant a process of converting a material, such as a biological sample, into a frozen solid that is relatively free of crystalline structures (e.g., water crystals). By relatively free of crystalline structures is meant that the number and/or size of crystalline structures (e.g., water crystals) within the sample frozen under high pressure conditions is reduced as compared to the number and/or size of crystaline structures produced by freezing a similar sample while the sample is not under high pressure conditions, e.g., by at least about two fold, such as by at least about four fold and including by at least about ten fold.

In certain embodiments, a “high pressure frozen sample” of the invention is one wherein the liquid component of the sample has been converted to a solid form, under high pressure, with a reduced formation of number and/or size of ice crystals as compared to a similar sample frozen at ambient pressure.

Freezing occurs by the removal of heat from the sample, wherein a sample is cooled. In certain embodiments, freezing of a sample is achieved by changing the temperature of the sample from a first temperature to a second temperature. In certain embodiments, the first temperature ranges from about 473 K to about 233 K, such as from about 350 K to about 252 K, such as from about 300 K to about 273 K, and the second temperature ranges from about 273 K to about 0 K, such as from about 225 K to about 77 K, while under pressure.

Freezing of the sample may be acheived using any convenient protocol, e.g., via application of a freezing (e.g., cryogenic) medium to the sample, where the freezing medium may be applied to the sample, for instance, by using the specific protocols detailed below. Although the freezing can take place over a prolonged period of time, in certain embodiments, it occurs rapidly, for instance, in a range of about 1 millisecond to about 1 second, from about 3 milliseconds to about 30 milliseconds, such as from about 7 milliseconds to about 15 milliseconds, e.g., about 10 milliseconds.

Freezing can take place either with or without the addition of agents such as cryoprotectants (for instance, glycerol, dimethyl sulfoxide, polyvinyl pyrridole, sucrose, antifreeze proteins and glycoproteins and the like) that suppress the formation of crystals. Although freezing can take place with the addition of agents that suppress the formation of crystals, such agents can be toxic to or otherwise adversly effect the sample. In certain embodiments, the freezing process occurs without the addition of cryoprotectants or with cryoprotectants that do not adversely affect the sample.

Freezing can take place either with or without the addition of agents such as baroprotectants (for instance trimethyl amine oxide, TMAO) that suppress injury to biological systems due to exposure to high pressure. Although freezing can take place with the addition of agents that suppress injury due to high pressure, such agents can be toxic to or otherwise adversly effect the sample. In certain embodiments, the freezing process occurs without the addition of baroprotectants or with baroprotectants that do not adversely affect the sample.

Any sample can be thawed in accordance with the methods of the invention. For instance, the methods are suitable for use with a biological sample, such as an organ, tissue, or cell sample. In certain embodiments, the cell sample includes one or more gamete cells, for instance, spermatozoa or ova. In certain embodiments, a sample includes multiple cells, for instance, embryonic stem cells. In certain embodiments, a sample includes a multicellular organism, for instance, an embryo. In certain embodiments, the sample is a viable biological sample. In certain embodiments, the sample may be one or more cells (e.g., a cell, a gamete cell, stem cell, neuronal cell or the like) that has been associated with a substrate, for instance, a glass, silicon or electronic chip. Further, the one or more cells may be arranged in a circuit configuration and may interact with other circuitry components to form a circuit that is capable of transferring a current or other signal from one point on the chip to another. In certain embodiments, the methods of the invention are characterized in that they are performed in such a manner and under conditions that preserve or maintain the viability of a biological sample. By “viability” of a biological sample is meant that the biological sample and/or one or more of its components maintains its ability to function, divide, differentiate, grow or otherwise live.

Another aspect of the invention is an apparatus for both freezing and thawing a sample under pressure. In certain embodiments, an apparatus of the invention is characterized in that it is configured for both freezing and thawing a biological sample under high pressure and in a manner sufficient to maintain the structural integrity and/or viability of the biological sample.

An apparatus of the invention includes a chamber, a pressure modulator and a temperature modulator. The chamber includes an interior that is configured for holding a sample. For instance, where the pressure modulator includes two opposing surfaces (e.g. anvils) the chamber may be a cavity created between the two surfaces (e.g., anvils). Additionally, the chamber may be the interior of a sample holding element that is adapted for holding a sample, for instance, a biological sample, and configured for being associated between the two surfaces (e.g., anvils) of the pressure modulator. For instance, the sample holding element may be a gasket, foil, membrane, or the like. The sample holding element may be fabricated out of any material (e.g., metal) so long as it is capable of associating with the opposing surfaces of the pressure modulator in a manner sufficient to withstand a high pressure generated by the pressure modulator. In certain embodiments, the sample holding element is a hard metal foil associated between two opposing surfaces and adapted for both holding a sample and supporting the contact point of the two surfaces. In certain embodiments, the chamber may include a hydrostatic fluid.

The pressure modulator may be of any configuration so long as it is adapted for generating a pressure difference between the interior and the exterior of the chamber. Accordingly, the pressure modulator modulates the pressure of the interior of the chamber. In certain embodiments, the pressure modulator is a high pressure modulator capable of generating a high pressure within the chamber. By “high pressure” is meant a pressure in the range of about 200 atm to about 4000 atm, for instance, about 1800 atm to about of 2800 atm, or about 2000 atm to about 2200 atm. In certain embodiments, the pressure modulator is capable of generating a high pressure rapidly, for instance, in about 1 millisecond to about 30 milliseconds, such as from about 7 milliseconds to about 15 milliseconds, e.g., about 10 milliseconds.

In certain embodiments, the pressure modulator may include two opposing surfaces and a force generating mechanism (e.g., a compression mechanism). For instance, the pressure modulator may include two opposing surfaces (e.g., anvils) that are configured to form a chamber and/or associate with a sample holding element in a manner so as to form a chamber and are additionally operatively connected to a force generating mechanism in a manner sufficient to allow the two opposing surfaces to be compressed one toward the other which thereby generates a high pressure within the chamber. The force generating mechanism which is operatively connected to the two surfaces may include one or more lever arms, screws, hydraulic systems or the like that are configured for being tightened or pressurized and thereby compressing the two opposing surfaces toward one another. In certain embodiments, the operative connection is such that it generates a uniaxial force that is applied to the base of the opposing surfaces thereby compressing the surfaces together and consequently generating a high pressure within the chamber.

As will be described in greater detail herein below, in certain embodiments, the two opposing surfaces of the pressure modulator may be anvils. By “anvil” is meant a hard, fixed surface that is operatively connected with a force generating mechanism and configured for being compressed against a second hard, fixed surface and thereby generating a high pressure at the region of contact between the two surfaces. The anvils may be fabricated of any material capable of being compressed and withstanding the generation of a high pressure due to said compression without fracturing. Although the anvils may be fabricated from any suitable material, in certain embodiments, one or more of the anvils is a material with a high thermal conductivity. By high thermal conductivity material is meant a material with a thermal conductivity ranging between about 300 W m⁻¹ K⁻¹ to about 5400 W m⁻¹ K⁻¹, about 895 W m⁻¹ K⁻¹ to 2300 W m⁻¹ K, or about 1000 W m⁻¹ K⁻¹ to 2000 W m⁻¹ K. For instance, the anvils may be diamonds, sapphires, rubies, emeralds, or other precious or non-precious gem quality stones. Accordingly, a suitable device of the invention may be configured as a diamond anvil cell.

In certain embodiments, the anvils include at least one precious gemstone, for instance, a flawless diamond or other high quality gem stone, with between about 10 to about 20 facets, about 13 to 17 facets, or about 16 facets. The weight of a gemstone anvil may vary, but is typically about ⅛ to about 2 carats, about ¼ to about ⅝ carat, or about ⅓ carat. The tip of the gemstone anvil, for instance, a diamond, may be cut, ground and/or polished so as to form a desired surface shape, for instance, a hexadecagonal surface.

The temperature modulator may be of any configuration so long as it is adapted for modulating the temperature of the interior and/or exterior of the chamber. By “modulating the temperature of the interior and/or exterior of the chamber” is meant that the temperature modulator is capable of changing the temperature of the interior or exterior of the chamber from a first temperature to a second temperature. Accordingly, the temperature modulator controls the temperature of the interior of the chamber and is configured for changing the temperature within the chamber along a broad range of temperatures. In certain embodiments, the temperature modulator is configured for modulating the temperature of the interior in a range that includes a temperature that is below the freezing point of water to a temperature that is above the freezing point of water.

Specifically, in certain embodiments, the temperature modulator is configured for modulating the temperature within the chamber over a range from about 0 K to about 473 K, about 40 K to about 350 K, or about 125 K to about 274 K. Hence, the temperature modulator is adapted for modulating the temperature within the chamber and thereby modulating the temperature of a contained sample. In certain embodiments, the temperature modulator is configured for rapidly heating or cooling the chamber, for instance in a about 1 millisecond to about 250 milliseconds, such as from about 2 milliseconds to about 150 milliseconds, such as from about 3 milliseconds to about 100 milliseconds, such as from about 5 milliseconds to about 50 milliseconds, such as from about 7 milliseconds to about 20 milliseconds, e.g., about 10 milliseconds.

In certain embodiments, the temperature modulator includes a heating element. The heating element may be any means capable of generating and causing the transference of a high temperature (i.e., heat) to the interior of the chamber. For instance, a heating element may include a fluid, such as a gas or liquid that contacts the pressure modulator and/or chamber and thereby warms it. In certain embodiments, the heating element includes a helium gas or water that is heated and contacted with one or more of the opposing surfaces, e.g., anvils, of the pressure modulator.

In certain embodiments, the heating element is configured for contacting the pressure modulator with both a heated helium gas and heated water. Accordingly, in these embodiments, the heating element is configured for heating the exterior components of the apparatus (e.g., the pressure modulator, anvils, sample holding element, gasket, etc.) which in turn transfers heat to the inside of the chamber and thereby warms the sample. In certain embodiments, the heating element may add heat directly to the inside of the chamber, for instance by means of a resistive electrical element located inside the sample chamber. In certain embodiments, the heating element may add heat directly to the anvils, for instance by passing electrical current through anvils that are made of electrically conductive or semiconductive material, or by heating the anvils and/or the sample by means of magnetic inductive heating. In certain embodiments, the heating element may operate by irradiating the sample and/or the anvils with light or microwave energy or other electromagnetic energy which is absorbed by the material of the sample and/or the anvils. In certain embodiments, the heating element may operate by means of adiabatic magnetization of the anvils and/or the sample. Accordingly, in these embodiments, the heating element is configured for heating the interior or interior components of the apparatus (e.g., of the sample, anvils, sample holding element, gasket, etc.)

In certain embodiments, the temperature modulator includes a cooling element. The cooling element may be any means capable of withdrawing heat from the interior of the chamber. For instance, a cooling element may include a fluid, such as a gas or liquid that contacts the pressure modulator and/or chamber. In certain embodiments, the cooling element includes a cryogenic solution, for instance, liquid nitrogen that is contacted with one or more of the opposing surfaces, e.g., anvils, of the pressure modulator. Accordingly, in these embodiments the cooling element is configured for cooling the exterior components of the apparatus (e.g., the pressure modulator, sample holding element, etc.) that in turn cool the inside of the chamber and thereby freeze the sample, for instance, under high pressure. In certain embodiments, the cooling element is configured for cooling the interior components of the apparatus. For instance, the cooling element may remove heat from the sample or from the anvils by means of adiabatic demagnetization of the anvils, or of the sample. In certain embodiments, the cooling element may operate by means of laser or optical cooling, as in the manner of the Los Alamos Solid State Optical Refrigerator. In this embodiment, the anvils may be made of glass doped with Ytterbium, or other suitable compounds.

An apparatus of the invention may have any configuration so long as it includes a chamber for holding a sample that can withstand a high pressure and includes both a means of generating a high pressure within the chamber and a means for rapidly transferring heat to and from the chamber (i.e., for thawing or freezing a sample). Accordingly, an apparatus of the invention can be fabricated from a wide variety of materials, as is known in the art, but should be fabricated out of materials that can withstand both high pressure and rapid changes in extreme temperatures. The construction and operation of anvil-type high pressure chambers are well known in the art and disclosed in the publications which are expressly incorporated in their entirety herein by reference below.

In certain embodiments, the devices of the invention are adapted for both rapidly transferring heat to and from a chamber, for instance, a sample holding element under high pressure, for thawing and/or freezing a sample, in a manner sufficient to maintain the structural integrity and/or viability of the sample. An apparatus of the invention is configured for both supplying heat to and withdrawing heat from a sample, for instance, a biological sample, that is held within a pressure chamber (e.g., by a sample holding element). In certain embodiments, an apparatus of the invention is configured so as to both cryogenically cool and warm a sample sequentially, one or more times (e.g., repeatedly), in a controlled manner that allows for the precise control of the pressure and temperatures generated, as well as the time period during which those pressures and temperatures are generated.

To better understand an apparatus of the invention, a specific embodiment of a high pressure chamber in operative communication with two opposing anvils, a gasket sample holder and a temperature modulator is set forth herein below. Although the following description is set forth with reference to a particular embodiment of an apparatus of the invention for use in accordance with the methods of the invention, it is to be understood that an apparatus of the invention and its components can have a variety of configurations as will be understood by those of skill in the art.

As can be seen with reference to FIG. 1, in certain embodiments, an apparatus of the invention (100) contains a pressure modulator that includes both a force generating mechanism (e.g., a compression mechanism) (118) and two opposing elements configured as anvils (110 a and 110 b) (e.g., diamonds). The apparatus (100) further includes a sample holding element (116) (e.g., a gasket) with an interior that forms a chamber (not shown), which is configured for holding a sample. The apparatus (100) additionally includes a temperature modulator (130) configured for modulating the temperature of the chamber from a temperature that is below the freezing point of water to a temperature that is above the freezing point of water.

The compression mechanism (118) includes two mount plates (120 a and 120 b) that are operatively joined by two screws (122 a and 122 b). The anvils (110 a and 110 b) include a base (111 a and 111 b) and culets or tips (112 a and 112 b). The anvils (110 a and 110 b) are associated with the mount plates (120 a and 120 b) in such a manner that the base (111 a and 111 b) of each anvil contacts the mount plates (120 a and 120 b) and the tips (110 a and 110 b) of the anvils are parallel and face one another. The mount plates hold the anvils in a fixed position. Tips (112 a and 112 b) contact the two sides and the interior of the sample holding element (116). The juncture of the tips (112 a and 112 b) and the interior of the sample holding element (116) creates a chamber (not shown) within which a sample, for instance, a biological sample, can be held. Accordingly, within the created chamber an enclosed sample can be thawed and/or frozen in accordance with the methods of the invention, e.g., under pressure.

The pressure modulator is configured for modulating a pressure within the chamber. The compression mechanism (118) is operatively connected with the mount plates (120 a and 120 b) which are in turn associated with the base (111 a and 111 b) of anvils (110 a and 110 b) and is configured for being compressed and thereby compressing the tips (112 a and 112 b) of the anvils together. The force generated by the compression of the anvils (110 a and 110 b) together generates a pressure within the chamber. As shown, the two screws (122 a and 122 b) are configured for being tightened and thereby compressing the mount plates (120 a and 120 b) together. It is to be noted that although with respect to the illustrated embodiment, the compression mechanism is configured for being compressed by the tightening of one or more screws, this should in no way be construed as limiting the compression mechanism in that the compression could also be generated by one or more suitably configured lever arms.

The temperature modulator (130) includes a heating source (132), a cooling source (134), one or more delivery conduits (136) and one or more delivery mechanisms (138). The heating source (132) may be a fluid reservoir for containing and heating a fluid, such as a gas (e.g., helium) or liquid (e.g., water). The cooling source (134) may be a fluid reservoir for containing and cooling a fluid, such as a cryogenic fluid (e.g., liquid nitrogen). The heating or cooling source may further be connected to an electrical source.

The delivery conduit (136) is configured for delivering a heated or cooled fluid to the delivery mechanism (138). The delivery conduit may be connected to only the heating source, to only the cooling source, or to both. Accordingly, the delivery conduit may be one or a plurality of tubes, pipes, or the like. The delivery conduit may be fabricated from any material capable of transporting fluids and withstanding extreme temperatures. For instance, the delivery conduit may be fabricated from plastic, glass, metal or the like.

The one or more delivery mechanisms (138) is configured for receiving the heated or cooled fluid from the one or more delivery conduits and delivering the received fluid to the apparatus of the invention in a manner sufficient to heat or cool the other components of the device, for instance, the anvil(s) (110 a and/or 110 b) and/or the sample holding element (116). Specifically, the delivery mechanism, as shown, may be configured for contacting one or more of the pressure modulators (e.g., one or more anvils thereof) and the sample holding element (116) with a heating or cooling fluid of the invention and thereby heating or cooling the chamber.

Although with respect to the illustrated embodiment, the temperature modulator is configured for heating or cooling a sample by contacting a chamber containing the sample and thereby heating or warming the sample, it is to be noted, that other configurations for producing a high pressure chamber and/or heating and/or cooling the sample may also be provided as is well known in the art and described above.

In another embodiment, the anvil includes at least one gem stone, for instance, a diamond, and a post, for instance, a metal post. In certain embodiments, the gem stone anvil (e.g., diamond) and the post interact with a sample containing element to produce a sample or pressure chamber. For example, as can be seen in reference to FIG. 2, in one embodiment, a sample chamber may include a disk (e.g., a metal disk, such as copper) (203) for containing a sample. The disk may contain a depression (201) in which the sample is placed. This disk may be placed between a single diamond anvil (202) and a metal post (208) to produce a pressure chamber in the depression (201). The diamond anvil (202) is contacted by a pressure plate (209) through mating surface (210) and is configured to contact the sample and cover the depression (201) in the disk (203) thereby enclosing the sample in the depression (201) in the disk. The sample may thereby be sealed inside the depression (201) with a pressure tight seal by applying uniaxial compressive forces to surfaces (207) and (206) of the pressure plate (209) and the post (208).

In accordance with this embodiment, the post may contain a hole (205). This hole may further contain a fluid. For instance, a liquid or a gas (e.g. helium) may be contained within the hole (205) of the post (208). The bottom surface (204) of the disk (203) may be positioned to cover the hole (205) in the post (208). The sample chamber may be sealed against the bottom surface of the diamond anvil (202) by applying a moderate uniaxial compressive force to surfaces (206) and (207), which application of these compressive forces also creates a pressure tight seal between the bottom surface (204) of the disk (203) and the fluid volume of the hole (205). Pressure may be applied to the sample by means of a fluid (e.g. helium gas) in the hole in the post. For example, when pressure is applied via the fluid in the hole (205), the pressure is transmitted to the bottom (204) of the metal disk (203) containing the sample. The pressure may then deform the bottom (204) of the metal disk and pressurize the sample in the sample chamber (201). After pressurizing the sample in the sample chamber, the temperature modulating fluid (e.g. helium gas and/or water) may be applied by means of a fluid delivery manifold (211) to the outside of the diamond anvil (202), the metal disk (203) and/or the metal post (208). In addition, heat may also be added to the sample in the sample chamber (201) by irradiating the sample chamber with light (212) or other electromagnetic radiation from a source (213), which may pass through the diamond anvil (202) into the sample chamber (201).

A feature of the temperature modulator is that it is configured for both heating and cooling the chamber one or more times (e.g., repeatedly). Because of the rapid heat transfer characteristics of the system (e.g., the rapid heat transfer characteristics of one or more of the opposing surfaces of the pressure modulator that interact to form the chamber), the temperature modulator is configured for modulating the temperature of the interior of the chamber and thereby heating or cooling a sample contained therein by heating or cooling the exterior of the chamber. For instance, the temperature modulator is configured for modulating the temperature of the interior of the chamber in a temperature range that is below the freezing point of water to a temperature that is above the freezing point of water and vice-versa. Specifically, the temperature modulator is configured for modulating the temperature within the chamber from about 0 K to about 473 K, about 40 K to about 350 K, or about 125 K to about 274 K. The rate of modulation may be from about 0.1° C./min to about 5000° C./ms, from about 1° C./min to about 40° C./ms, from about 10° C./min to about 1° C./msec. Accordingly, in certain embodiments, the thawing or freezing of a sample occurs at a rate ranging from about 0.1° C./min to about 5000° C./millisecond.

In certain embodiments, an apparatus of the invention is useful for both thawing (e.g., heating) and freezing (e.g., cooling) of a sample under pressure, for instance, high pressure, in a manner sufficient to prevent or reduce in size and/or number the formation of ice crystals within the sample normally caused by a thawing or freezing process that is not performed under high pressure. Where the sample is a biological sample, for instance a cell or tissue sample or microorganism, the thawing and freezing occur without substantially disrupting the structural integrity of the biological sample. Additionally, where the sample is a viable biological sample, the thawing and freezing occur in a manner sufficient to maintain the viability of the sample.

In one aspect, the subject invention is directed to a method of freezing and/or thawing a sample under pressure, for instance, high pressure. A sample, for instance, a biological sample, is obtained. The sample may be any sample the analysis and/or manipulation of which is desired. In certain embodiments, the sample may be obtained from any suitable source in any manner sufficient to preserve the integrity of the sample, as is well known in the art. Where the sample is a biological sample it may be obtained from a suitable organ and/or tissue of interest. For instance, the sample may be a blood sample collected from a subject's veins via venipuncture, the sample may be an epidermal sample collected via skin grafting or the sample may be a tissue sample collected from some other organ (e.g., a liver, kidney, lungs, heart, brain or the like), or the sample may be spermatozoa or ova, or the sample may be an embryo or stem cells derived from an embryo.

In one embodiment, the tissue to be observed and/or analyzed and/or manipulated is from an organ (e.g., a brain) and the tissue of interest (e.g., neural tissue) is excised from that organ in a manner sufficient to preserve the viability of the sample. Accordingly, the organ (e.g., brain) from which the tissue (e.g., neural tissue) is to be harvested may first be put into a state of cold but not frozen suspended animation (e.g., at a temperature between 273 K and 283 K) and then carefully sliced in a manner to reduce damage to the tissue sections collected, as is well known in the art. The sliced sections may range in thickness from about 10 μm to about 300,000 μm, such as from about 20 μm to about 1000 μm, e.g., from about 200 μm to about 400 μm. The tissue (e.g., neuronal cells) collected may then be placed into a chamber of a device of the invention. For instance, a chamber formed between the two opposing surfaces of the pressure modulator or a chamber formed from the interior of a suitable sample holder (e.g., a gasket which is then placed between the two opposing surfaces of the pressure modulator).

The sample is placed in between the two opposing surfaces of the pressure modulator and the opposing surfaces are aligned and manipulated so as to generate a pressure within the chamber, for instance, a high pressure. The high pressure may be generated by manipulating the force generating mechanism (e.g., suitably configured lever arm(s) or screw(s) or hydraulic system) of the pressure modulator in a manner sufficient to cause the tips of the two opposing surfaces to move toward, contact, and be compressed against one another and/or the sample and/or the gasket, which thereby generates a high pressure within the chamber.

A high pressure is generated within the chamber and the temperature modulator is engaged to apply a cooling fluid to the exterior of the chamber (e.g., to the opposing surfaces of the pressure modulator and/or the exterior of the sample holder) in a manner sufficient to cause the freezing of the sample with minimal to no ice crystal formation within the sample (e.g., both within and between the cells of the sample). The freezing of the sample may take place rapidly, as described above, and in a manner such that the sub-cellular structures and their positioning remains unaffected (e.g., by ice crystal formation) and the cell to cell alignment within the tissue remains intact. In certain embodiments, the freezing of a biological sample takes place in a manner such that the chemical, biochemical and molecular processes within the biological sample cease.

A frozen sample may be thawed in a manner sufficient to maintain the structural integrity and/or viability of the sample. Accordingly, to thaw a frozen sample, the sample is placed within a chamber of the apparatus (if not already therein). For instance, the sample may be placed within a sample holder (e.g., a gasket, washer, embossed metal plate or the like) and the sample holder may then be placed between the opposing surfaces of the pressure modulator. The opposing surfaces of the pressure modulator (e.g., anvils) are then aligned and brought together so as to enclose the chamber. Once the chamber is enclosed between the two opposing surfaces the pressure modulator is manipulated so as to generate a pressure within the chamber, for instance, a high pressure. The high pressure may be generated by manipulating the force generating mechanism (e.g., a suitably configured lever arm(s) or screw(s) or hydraulic system) of the pressure modulator in a manner sufficient to cause the two opposing surfaces to move toward one another thereby generating a high pressure within the chamber.

A high pressure is generated within the chamber and the temperature modulator is then be engaged to apply a heating fluid to the exterior of the chamber (e.g., to the opposing surfaces of the pressure modulator and/or the exterior of the sample holder) in a manner sufficient to cause the thawing of the sample with minimal to no ice crystal formation within the sample (e.g., both within and between the cells of the sample). The thawing of the sample may take place rapidly, as described above, and in a manner that the sub-cellular structures and their positioning remains relatively unaffected (e.g., due to the melting of fluid components and/or recrystallization of ice within and between the cells of the sample) and the cell to cell alignment within the tissue remains intact.

Once thawed the cells of the sample maintain their viability and continue their typical cellular processes. In certain embodiments, the thawed sample may be manipulated in one or more ways. For instance, the sample may be physically or chemically manipulated. Once manipulated the sample may then be refrozen in accordance with the methods of the invention to produce a frozen sample with an intact structural integrity and/or viability following the manipulation. In certain embodiments, the thawed or frozen sample may be imaged and/or observed. A suitable apparatus for use in imaging and/or manipulating a sample in accordance with the methods of the invention is set forth in the Applicants' co-pending application entitled Methods and Device for Imaging and Manipulating a Biological Sample. Attorney Docket Number [BIOT009US2], which is herein incorporated by refrence.

Once thawed the sample may then be refrozen in a manner sufficient to maintain the structural integrity and the viability of the sample. In this way, this process may be performed repeatedly over several cycles of freezing and thawing under pressure and in a manner sufficient to maintain the structural integrity and/or viability of the sample.

Additionally, the methods described herein are also useful for storing a viable biological sample that maintains its structural integrity and viability so as to be thawed at a later time.

For instance, the methods herein described are useful in maintaining and storing living cells contained on silicon chips. Mammalian cells, such as nervous system cells (e.g., neurons) may be associated with a silicon microchip so as to form a biological circuit. For instance, in certain embodiments, one or more cells (e.g., a cell, a neuron, a gamete cell, stem cell, or the like) may be associated with a substrate, for instance, a glass, silicon or electronic chip so as to form a biological circuit or other useful device. For example, the one or more cells may be arranged in a circuit configuration and may interact with other circuitry components to form a circuit that is capable of transferring a current or other signal from one point on the chip to another. In another example, the one or more cells may be arranged in a way to allow the cells to be stimulated and/or observed by optical, electronic or other means.

The association of a biological cell with a substrate so as to form a microchip that contains biological components is well known in the art and disclosed in following references, which are hereby incorporated by reference in their entirety for their teaching on the production and use of biochips. The Neurally Controlled Animat: Biological Brains Acting with Simulated Bodies, Thomas B. DeMarse, Daniel A. Wagenaar, Axel W. Blau and Steve M. Potter Autonomous Robots v.11 n.3 p. 305-310 (November 2001). Noninvasive neuroelectronic interfacing with synaptically connected snail neurons immobilized on a semiconductor chip, Günther Zeck and Peter Fromherz PNAS Aug. 28, 2001, vol. 98 no. 18 p. 10457-10462. Engineering a biospecific communication pathway between cells and electrodes, Joel H. Collier and Milan Mrksich PNAS Feb. 14, 2006, vol. 103 no. 7 p. 2021-2025. Closing the Loop: Stimulation Feedback Systems for Embodied MEA Cultures, S. M. Potter, D. A. Wagenaarand T. B. DeMarse, in: Advances in Network Electrophysiology Using Multi-Electrode Arrays, M. Taketani and M. Baudry (Eds.), Springer (2005). For instance, cortical neurons from a suitable organism may be dissociated and cultured on a surface containing a grid of electrodes (multi-electrode arrays, or MEAs) capable of both recording and stimulating neural activity.

Such microchips containing biological circuits or other devices and sensors may be useful in studying the behavior of neurons, in analyzing the information processing functions of particular neurons or samples of neural tissue, in drug screening systems, in systems for detecting environmental pathogens or toxins, in chemical sensors (artificial noses), in the development of neural prostheses, in the generation of organic computers using living neurons, and in other applications. Accordingly, the methods of the invention are useful for cryogenically storing neurons or other cells before their employment in such a biochip and/or for cryogenically storing the neuron or other cell containing biochips once they have been fabricated. Hence, the methods herein disclosed are useful in both studying the effects of and implementing cryogenic storage of biochips containing neurons or other cells.

In another aspect, the present invention is directed to a computer program that may be utilized to carry out the above steps. One or more of the steps including: the placement of a sample into a chamber, the alignment of the opposing surfaces of the pressure modulator, the enclosing of the chamber, the generation of a force, the modulation of the pressure within the chamber, the modulation of the temperature within the chamber, the placement of the sample and/or chamber components on a stage, the monitoring (e.g., of temperature and pressure) of the sample, the removal of the sample from the chamber, in accordance with the invention, may all be done automatically under computer control, that is, with the aid of a computer. The computer may be driven by software specific to the methods described herein. Examples of software or computer programs used in assisting and conducting the present methods may be written in any convent language, e.g. Visual BASIC, FORTRAN and C++ (PASCAL, PERL or assembly language). It should be understood that the above computer information and the software used herein are by way of example and not limitation.

Programming according to the present invention, i.e., programming that allows one to carry out the methods of the invention, as described above, can be recorded on computer readable media, e.g., any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media such as floppy discs, hard disc storage media and magnetic tape; optical storage media such as CD-ROM and DVD; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.

In certain embodiments, a processor of the subject invention may be in operable linkage, i.e., part of or networked to, the aforementioned apparatus, and capable of directing its activities. A processor may be pre-programmed, e.g., provided to a user already programmed for performing certain functions, or may be programmed by a user.

Thus, in certain embodiments, the programming is further characterized in that it provides a user interface, where the user interface presents to a user the option of selecting among one or more different, including multiple different, rules for individually controlling the steps of the methods herein disclosed. A processor may be remotely programmed by “communicating” programming information to the processor, i.e., transmitting the data representing that information as electrical signals over a suitable communication channel (for example, a private or public network). Any convenient telecommunications means may be employed for transmitting the programming, e.g., facsimile, modem, Internet, LAN, WAN or other network means, wireless communication, etc.

EXAMPLES Modeling in Software the Cooling and Warming of a Sample

High pressure freezing is used by electron microscopists freeze biological samples with the formation of few or no ice crystals, and/or ice crystals of greatly reduced size, thus preserving the fine structure of those samples for later imaging. Success in freezing without ice crystal formation, for aqueous samples thicker than about 10 to 20 microns, may depend on the timed application of sufficiently high pressure and sufficiently rapid cooling to the biological sample being frozen. Use of chemical cryoprotectants may relax these conditions to some degree, but in certain embodiments, little or no cryoprotectant need be used, for instance, in circumstances where the use of cryoprotectant chemicals may be toxic to viable biological samples. Thus, the present methods and devices may be configured for reversibly thawing and freezing viable biological samples, without sacrificing viability and without the use of cryoprotectant chemicals. In certain embodiments, a device of the invention may be configured to thaw a biological sample under high pressure with the same rapidity that the sample was frozen.

The heat transfer characteristics of a device of this patent were modeled, using COMSOL Multiphysics version 3.2b software. The materials of the device were alloy steel, copper, water, and diamond. Images of the temperature distribution at all relevant points in the device, for various times after commencement of warming or cooling, were generated by the software.

FIGS. 3 through 5 show the predicted temperature distribution in the sample cell of a device of the invention during cooling. The figures are side view cross sections, and the sample cell has axially symmetric geometry. The sample cell also has bilaterally symmetric geometry, about the plane perpendicular to the device axis passing through the center of the sample cell. Accordingly, FIGS. 3 through 5 show only one quarter (the upper right quadrant) of the complete cross section. Therefore in the figures the leftmost edge is the centerline axis of the device, and the bottom edge cuts through the center of the sample and the copper gasket that holds the sample. The diamond anvil cooling plate is 1.2 mm thick by 6 mm in diameter, and is represented in the figures by a rectangle 1.2 mm high by 3 mm wide. The alloy steel post holding the diamond anvil cooling plate is represented by the rectangular region immediately above the diamond anvil cooling plate. Initial temperature conditions for the model at time t=0 are 275 K throughout the volume of the device, and 77 K at the outer surface of the device. The temperature scale is shown by the vertical colored bar at the right, with blue representing 75 K and red representing 275 K. Times shown are 1 millisecond, 10 milliseconds, and 100 milliseconds.

FIGS. 6 through 8 show the predicted temperature distribution in the sample cell of a device of the invention during warming. Device geometry and time intervals are the same as in FIGS. 3 through 5. At time t=0, the entire volume of the device is at 77 K, with the outside surface of the device warmed to 305 K.

Also generated were predicted temperature versus time curves for the point most central in the sample, which is the point in the sample most slowly cooled or warmed. FIG. 9 shows the predicted temperature in the center of the sample, for the case of cooling down in a device of this application. FIG. 10 shows the predicted temperature in the center of the sample, for the case of warming up in a device of this patent. Both figures show temperature versus time for times t=0 to t=3 seconds.

Comparing these images and the cooling and warming curves shows that the cooling rates achieved by the device of this patent are comparable to those obtained by electron microscopists in preparing their samples. Furthermore, the time to warm (e.g. thaw) the sample, using the device of this patent, is substantially equivalent to the cooling times of the device of this patent. This is what we want to achieve in this particular embodiment of a device of this patent.

The images and curves presented indicate the temperature versus time for points in the sample during cooling and warming, for instance, in the center of the sample. Points near the center of the sample will cool and warm most slowly, compared to points near the edges of the sample. The methods and devices of the invention are configured to rapidly cool and/or warm the entire sample with little or no ice crystal formation and while maintaining the structural integrity and/or viability of the sample.

Experimental Evaluation of the Survival of HP Frozen Rat Brain Tissue

The survival of rat brain tissue when exposed to low temperatures and high pressures was evaluated. In one procedure, a young female Sprague-Dawley rat, 273 grams, was anesthetized with 0.35 ml KAX (a mixture of ketamine, xylazine and acepromazine) intraperitoneally, weighed and cannulated in the femoral artery. The femoral vessel was opened and the animal was perfused with 45 ml of ice cold bicarbonated Hextend, until the hematocrit was reduced to less than one. The brain was then removed and sectioned into quarters and the four quarters treated as follows: 1) control, held for 15 minutes at 2° C.; 2) pressurized to 2177 atm for 5 minutes while at 2° C., then depressurized; 3) frozen to −196° C. at ambient (1 atm) pressure for 5 minutes and then thawed; and 4) pressurized to 2177 atm (221 MPa), then frozen to −196° C. over 5 minutes, held at this temperature and pressure for 5 minutes, then thawed to 2° C. over 15 minutes, then depressurized. Brain tissue in each case was kept in ice-cold bicarbonated Hextend saturated with BioBlend®¹³, a gas mixture consisting of 95% oxygen and 5% carbon dioxide.

After these steps, the brain tissue was cut into 200 μm thick coronal slices using a Vibratome®. While being sliced, the tissue was held at 2° C. while it was bathed in artificial cerebrospinal fluid (ACSF) bubbled with BioBlend. After slicing, the tissue samples were allowed to rest for about 45 minutes at room temperature, while bathed in ACSF bubbled with BioBlend. Slices were then stained by immersion for 30 minutes in room temperature BioBlend-bubbled Hextend to which the two-component Molecular Probes L-3224 Live/Dead stain was added. The L-3224 Live/Dead stain stains the cytoplasm of living cells green and the nuclei of dead cells red. Concentration of the calcein-AM component was 4.5 μM and concentration of the ethidium homodimer component was 6.7 μM.

Immediately after staining, the tissue was imaged using a Zeiss Meta-510 Confocal Laser Scanning Microscope (LSM). The microscope was fitted with a 40× water immersion objective and the illumination source was a 488 nm argon-ion laser. Three-dimensional stacks 236 μm high consisting of 50 2-D images measuring 650 μm×650 μm were acquired. The resulting 3-D image data sets were imported into the Zeiss LSM image browser program and individual 2-D slices were selected as representative images of the 3-D data sets. This live/dead viability assay showed that, when compared to the control, the pressure only tissue showed comparable viability, and the pressure frozen tissue showed much less viability.

FIGS. 11 through 13 illustrate the results of the different treatment of the sections. FIG. 11 illustrates the treatment of the control sample. As can be seen with reference to FIG. 11, the sample shows very few red spots interspersed on a strongly green colored cytoplasm matrix. Ropey structures which are the intact capillaries of the vascular bed can be seen threading through the image.

As can be seen with reference to FIG. 12, the tissue sample treated with pressure only shows less intense green color, but still very few red spots. The capillaries are clearly visible. Accordingly, the application of 2177 atm pressure to ice cold brain tissue for 5 minutes does not compromise tissue viability, when compared to control tissue samples (see FIG. 11).

Images of brain tissue frozen to −196° C. at ambient pressure were not obtained because samples frozen without the application of high pressure were so delicate and friable that it was impossible to slice them on the Vibratome without causing the tissue to fall apart.

As can be seen with reference to FIG. 13, the pressure frozen and thawed sample shows much less green cytoplasm background, many more red spots and no visible capillary structures.

In another procedure, the survival of rat brain tissue when thawed after rapid high pressure freezing was evaluated. In this procedure a young female Sprague-Dawley rat of 243 g was anesthetized, cannulated and perfused with ice cold bicarbonated Hextend until the hematocrit was reduced to less than one. The brain was then removed and sliced into 200 μm thick coronal slices. The slices were then soaked in bicarbonated Hextend to which glycerol had been added to a concentration of 20% by volume. Slices were then quickly pressure frozen at 2100 atm in a Bal-Tec HPM-010 high pressure freezing machine and stored in liquid nitrogen for several days. Cooling from room temperature to −196° C. took about 10 milliseconds in the Bal-Tec machine.

After storage in liquid nitrogen for several days, slices were rewarmed at 1 atm pressure by immersing them for about 45 minutes in Hextend at 20° C. bubbled with BioBlend. Slices were then immediately stained with L-3224 Live/Dead stain and imaged with the Zeiss Meta 510 Confocal Laser Scanning Microscope (LSM) fitted with a 20× air objective. Illumination was with the 488 nm argon-ion laser. Three dimensional stacks 42 μm high consisting of 20 2-D images measuring 460 μm×460 μm (512×512 pixels) were acquired. The resulting 3-D image data sets were imported into the Zeiss LSM image browser program and individual 2-D slices were selected as representative images of the 3-D data sets. See FIGS. 14 and 15. As seen in comparisons between FIG. 11 (control) and FIGS. 14 and 15, brain tissue slices prepared, pressurized, frozen and thawed in this way showed good viability although structure was altered due to the formation of ice crystals while thawing. Structures resembling the capillaries are visible and viability of the tissue is comparable to that of control samples.

In a further procedure, the survival of rat brain tissue when thawed under high pressure is evaluated. In this procedure a young female Sprague-Dawley rat of 243 g is anesthetized, cannulated and perfused with ice cold bicarbonated Hextend® until the hematocrit is reduced to less than one, as described above. The brain is then removed and sliced into 200 μm thick coronal slices. The slices are then soaked in bicarbonated Hextend to which glycerol is added to a concentration of 20% by volume. Slices are then quickly pressure frozen at 2100 atm in a high pressure freezing and thawing apparatus of the invention and stored in liquid nitrogen for several days.

After storage the slices are quickly pressure thawed at 2100 atm and the temperature of the sample is changed from −196° C. to room temperature within about 100 msec in an apparatus of the invention. The slices are then immediately stained with L-3224 Live/Dead stain and imaged with the Zeiss Meta 510 Confocal Laser Scanning Microscope (LSM) fitted with a 20× air objective. Illumination is with the 488 nm argon-ion laser. Three dimensional stacks 42 μm high consisting of 20 2-D images measuring 460 μm×460 μm (512×512 pixels) are acquired. The resulting 3-D image data sets are imported into the Zeiss LSM image browser program and individual 2-D slices are selected as representative images of the 3-D data sets. Image data sets show that the tissue is intact and strong enough to be easily sliced into 200 μm thick slices. Structures resembling the capillaries are visible and viability of the tissue is comparable to that of control samples.

It is evident from the above discussion that the subject invention provides an important breakthrough in the preparing biological samples without destroying the structural integrity and/or viability of the sample. Specifically, the subject invention allows one to thaw and freeze a viable biological sample one or more times in a manner sufficient to maintain the structural integrity and viability of the sample. Accordingly, the subject invention represents a significant contribution to the art.

All publications and patents cited in this specification are herein incorporated by reference, in their entirety, as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

REFERENCES

The following references discuss the design, construction and operation of high pressure diamond anvil cells:

-   Field, The Properties of Diamond, Academic Press, New York City,     N.Y. (1979); Manghnani, et al., High-Pressure Research and Mineral     Physics, Terra Scientific Publishing Company, Tokyo, American     Geophysical Union, Washington, D.C. (1987); Homan, “Higher Pressure     in Science and Technology”, Mat. Res. Soc. Symp. Proc., vol. 22, pp     2939, et seq., Elsevier Science Publishing Company (1984); Vodar, et     al., High Pressure Science and Technology, Proceedings of the VIIth     International AIRTAPT Conference, Le Creusot, France, Jul. 30-Aug.     3, 1979, Pergamon Press, New York, N.Y.; and Ruoff et al, “Synthetic     Diamonds Produce Pressure of 125 GPa (1.25 Mbar)”, J. Mater. Res., 2     (5), 614-617, September/October 1987

The following references discuss the design, construction and operation of high pressure freezing machines, for the purposes of cryofixing biological samples as a step in preparing samples for imaging in the electron microscope:

-   D. Studer, W. Graber, A. Al-Amoundi and P. Eggli, A new approach for     cryofixation by high-pressure freezing, Journal of Microscopy v203     part 3 p 285-294 (September 2001); Hans Moor, Cryotechniques in     Electron Microscopy p 176-191 (1987); E. Shimoni and M. Muller, On     optimizing high-pressure freezing: from heat transfer theory to a     new microbiopsy device, Journal of Microscopy v192 part 3 p 236-247     (December 1998); D. Studer, W. Graber, A. Al-Amoundi and P. Eggli, A     new approach for cryofixation by high-pressure freezing, Journal of     Microscopy v203 part 3 p 285-294 (September 2001); D. Studer, S.     Zhao, W. Graber, P. Eggli and M. Frotscher, High Pressure Freezing     of Brain Tissue Slices, Microscopy and Microanalysis v12 supplement     2 (2006) 

1. A method, comprising: thawing a frozen biological sample under pressure in a manner sufficient to maintain structural integrity of said biological sample.
 2. The method of claim 1, wherein said thawing occurs in an enclosed chamber.
 3. The method of claim 1, wherein said biological sample comprises multiple cells.
 4. The method according to claim 3, wherein said biological sample comprises a tissue sample.
 5. The method according to claim 1, wherein said biological sample comprises a multicellular organism.
 6. The method according to claim 1, wherein said biological sample comprises one or more ova or spermatozoa.
 7. The method according to claim 1, wherein said biological sample comprises one or more embryos.
 8. The method according to claim 1, wherein said biological sample comprises one or more adult or embryonic stem cells.
 9. The method according to claim 1, wherein said biological sample comprises one or more cells associated with a substrate.
 10. The method of claim 1, wherein said biological sample is viable.
 11. The method of claim 10, wherein said thawing occurs in a manner sufficient to maintain viability of said sample.
 12. The method of claim 1, wherein said pressure ranges from about 200 atm to about 4000 atm.
 13. The method of claim 1, wherein said thawing has a duration ranging from about 1 millisecond to about 1 second.
 14. The method of claim 1, wherein said thawing comprises warming said sample to a temperature ranging from about 250 K to about 310° K.
 15. The method of claim 1, wherein said thawing occurs at a rate ranging from about 0.1° C./min to about 5000° C./ms.
 16. The method of claim 1, further comprising manipulating said sample after said sample is thawed.
 17. The method of claim 15, wherein said manipulating comprises physically manipulating said sample.
 18. The method of claim 15, wherein said manipulating comprises chemically manipulating said sample.
 19. The method of claim 15, further comprising freezing said sample in a manner so as to maintain the viability of the sample to produce a frozen viable sample following said manipulating.
 20. The method of claim 19, further comprising observing said frozen viable sample,
 21. The method of claim 20, further comprising, following said observing, thawing said frozen sample under pressure in a manner sufficient to maintain the structural integrity of said sample.
 22. An apparatus comprising: a chamber having an interior configured to hold a sample; a pressure modulator for modulating the pressure of said interior; and a temperature modulator for modulating a temperature of said interior from a temperature that is below a freezing point of water to a temperature that is above a freezing point of water. 23-40. (canceled) 